Dear Philip,
the ratios between the different deoxys depend on the polymerase. Previously
the taq preferentially used the deoxynucletides rather that the
dideoxynucleotides. This problem is reduced by using Amplitaq FS. Now to
your question, the ratios between the dideoxynucleotide and the
deoxynucleotide is in the BigDye terminator kit as follows:
ddA/dA= 0.004
ddc/dC= 0.004
ddG/dG=0.003
ddT/dT=0.007
Hope this is of any use to you
Dag Ahrén
Microbial Ecology, Lund University, Sweden
Phillip San Miguel <pmiguel at purdue.edu> skrev i
diskussionsgruppsmeddelandet:819s37$sne at net.bio.net...
> The problem: on the 3700 short products apparently are injected
> preferentially over longer products--thus one tends to get much stronger
> signal at the beginning of a read than towards the end. Stephen Lasky
> mentioned that shortening the sample injection time ameliorates this
> problem. I've done this, but still often see signal in the beginnning so
> high that it maxes out the sequence analysis software and I lose
> sequence towards the end because the signal is too weak.
> I hear from PE-ABI that the "rebalanced" Big Dye kits (that favor
> longer products) won't be available until Jan 2000. These probably just
> adjust the ratio of deoxys to teminators to favor deoxys. So I could get
> the same effect by adding extra nucleotides. PE won't say what
> concentrations nucleotides are in the Big Dye kit. Does any one know or
> want to take a guess? Most troublesome would be getting the dITP
> concentration in the right range, I guess.
> The maddening aspect of PE-Bio's tardiness in releasing this kit is
> that at GSAC it was mentioned the the "rebalanced" or "reoptimized" kit
> gave better results for the 377 as well.
>> Phillip San Miguel
> Purdue Genomics Center
>
